RRC ID 53695
Author Mok WW, Park JO, Rabinowitz JD, Brynildsen MP.
Title RNA Futile Cycling in Model Persisters Derived from MazF Accumulation.
Journal mBio
Abstract UNLABELLED:Metabolism plays an important role in the persister phenotype, as evidenced by the number of strategies that perturb metabolism to sabotage this troublesome subpopulation. However, the absence of techniques to isolate high-purity populations of native persisters has precluded direct measurement of persister metabolism. To address this technical challenge, we studied Escherichia coli populations whose growth had been inhibited by the accumulation of the MazF toxin, which catalyzes RNA cleavage, as a model system for persistence. Using chromosomally integrated, orthogonally inducible promoters to express MazF and its antitoxin MazE, bacterial populations that were almost entirely tolerant to fluoroquinolone and β-lactam antibiotics were obtained upon MazF accumulation, and these were subjected to direct metabolic measurements. While MazF model persisters were nonreplicative, they maintained substantial oxygen and glucose consumption. Metabolomic analysis revealed accumulation of all four ribonucleotide monophosphates (NMPs). These results are consistent with a MazF-catalyzed RNA futile cycle, where the energy derived from catabolism is dissipated through continuous transcription and MazF-mediated RNA degradation. When transcription was inhibited, oxygen consumption and glucose uptake decreased, and nucleotide triphosphates (NTPs) and NTP/NMP ratios increased. Interestingly, the MazF-inhibited cells were sensitive to aminoglycosides, and this sensitivity was blocked by inhibition of transcription. Thus, in MazF model persisters, futile cycles of RNA synthesis and degradation result in both significant metabolic demands and aminoglycoside sensitivity.
IMPORTANCE:Metabolism plays a critical role in controlling each stage of bacterial persistence (shutdown, stasis, and reawakening). In this work, we generated an E. coli strain in which the MazE antitoxin and MazF toxin were artificially and independently inducible, and we used this strain to generate model persisters and study their metabolism. We found that even though growth of the model persisters was inhibited, they remained highly metabolically active. We further uncovered a futile cycle driven by continued transcription and MazF-mediated transcript degradation that dissipated the energy derived from carbon catabolism. Interestingly, the existence of this futile cycle acted as an Achilles' heel for MazF model persisters, rendering them vulnerable to killing by aminoglycosides.
Volume 6(6)
Pages e01588-15
Published 2015-11-17
DOI 10.1128/mBio.01588-15
PII mBio.01588-15
PMID 26578677
PMC PMC4659464
MeSH Anti-Bacterial Agents / pharmacology DNA-Binding Proteins / genetics DNA-Binding Proteins / metabolism* Drug Tolerance Endoribonucleases / genetics Endoribonucleases / metabolism* Escherichia coli / drug effects Escherichia coli / enzymology* Escherichia coli / metabolism Escherichia coli Proteins / genetics Escherichia coli Proteins / metabolism* Fluoroquinolones / pharmacology Gene Expression Glucose / metabolism Oxygen / metabolism Promoter Regions, Genetic RNA Stability RNA, Bacterial / metabolism* Ribonucleotides / metabolism Substrate Cycling Transcription, Genetic beta-Lactams / pharmacology
IF 6.784
Times Cited 22
Prokaryotes E. coli